Publications by authors named "Pengxia Wan"

31 Publications

The Role of Elastase in Corneal Epithelial Barrier Dysfunction Caused by Pseudomonas aeruginosa Exoproteins.

Invest Ophthalmol Vis Sci 2021 Jul;62(9)

Department of Ophthalmology, The First Affiliated Hospital, Sun-Yat-sen University, Guangzhou, China.

Purpose: To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa.

Methods: Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1β levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro.

Results: Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ±  2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor.

Conclusions: Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.
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http://dx.doi.org/10.1167/iovs.62.9.7DOI Listing
July 2021

Utility of multi-parametric quantitative magnetic resonance imaging of the lacrimal gland for diagnosing and staging Graves' ophthalmopathy.

Eur J Radiol 2021 Aug 8;141:109815. Epub 2021 Jun 8.

Department of Endocrinology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510080, Guangdong Province, China. Electronic address:

Purpose: To explore radiological changes of the lacrimal gland (LG) in Graves' ophthalmopathy (GO) based on multi-parametric quantitative MRI and its clinical utility in LG diagnosis and activity in GO.

Methods: We enrolled 99 consecutive patients with GO (198 eyes) and 12 Graves' Disease (GD) patients (24 eyes) from July 2018 to June 2020. Clinical, laboratory, and MRI data were collected at the first visit. Based on clinical activity scores, eyes with GO were subdivided into active and inactive groups. T2-relaxation time (T2) and the absolute reduction in T1-relaxation time (ΔT1) were determined. After MRI and processing, we performed descriptive data analysis and group comparisons. Novel logistic regression predictive models were developed for diagnosing and staging GO. Diagnostic performance of MRI parameters and models was assessed by receiver operating characteristic curve analysis.

Results: LG in GO group had significantly higher T2 and ΔT1 values than the GD group [106.25(95.30,120.21) vs. 83.35(78.15,91.45), P<0.001, and 662.62(539.33,810.95) vs. 547.35(458.62,585.57), P = 0.002, respectively]. The GO group had higher T2 of LG indicating higher disease activity [110.93(102.54,127.67) vs. 93.29(87.06,101.96), P < 0.001]. Combining T2 and ΔT1 values of LG, Model I had higher diagnostic value for distinguishing GO from GD (AUC=0.94, 95 %CI: 0.89,0.99, P<0.001). Meanwhile, T2 of LG had higher diagnostic value for grading GO activity (AUC = 0.84, 95 %CI: 0.76,0.92, P<0.001).

Conclusions: Multi-parametric quantitative MRI parameters of the LG in GO were significantly altered. Novel models combining LG T2 and ΔT1 values showed excellent predictive performances in diagnosing GO. Furthermore, T2 of LG showed practical utility for staging GO.
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http://dx.doi.org/10.1016/j.ejrad.2021.109815DOI Listing
August 2021

Corneal Endothelium: A Promising Quantitative Index for Graves Ophthalmopathy Activity Evaluation.

Am J Ophthalmol 2021 Jun 6;230:216-223. Epub 2021 Jun 6.

Department of Ophthalmology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong Province, China. Electronic address:

Purpose: To investigate the corneal endothelium damage in Graves ophthalmopathy (GO) and its role as a promising quantitative index to evaluate GO activity.

Design: Cross-sectional study.

Methods: This study included 128 eyes of 64 patients with GO. All subjects underwent ophthalmologic examinations, including proptosis, tear break-up time (BUT), corneal fluorescein staining, and Schirmer test. Corneal endothelium was measured by noncontact specular microscope and ocular biometric parameters were measured by IOLMaster 700. Each eye was assigned a specific clinical activity score (CAS), then grouped as active (CAS ≥3 points) or inactive (CAS <3 points). Ocular parameters between the 2 groups were compared using generalized estimating equations accounting for inter-eye correlation, and receiver operating characteristic (ROC) curves were also obtained. Main outcome measures were parameters of corneal endothelium.

Results: Among the included eyes, 81 eyes had inactive GO and 47 eyes had active GO. Corneal endothelial cell morphology was altered in active GO compared with inactive GO. The coefficient variation of cell area (CV) was significantly higher in active GO compared with inactive GO (37.0 [34.4-41.2]% vs 33.9 [30.9-36.8]%, P = .001), and positively correlated with CAS (r = 0.322, P < .001). Moreover, CV showed a diagnostic capacity to differentiate the active eyes from inactive eyes. The area under the ROC curve was 0.705.

Conclusions: Active GO had morphologic changes in corneal endothelium compared with inactive GO. CV is a sensitive indicator to reflect corneal endothelial function, and has the potential to be adopted as a noninvasive, objective, and quantitative index for evaluating the activity status of GO patients.
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http://dx.doi.org/10.1016/j.ajo.2021.05.020DOI Listing
June 2021

Bacterial agents and changes in drug susceptibilities in cases of chronic dacryocystitis, Southern China.

Int Ophthalmol 2021 Jan 19;41(1):1-10. Epub 2020 Aug 19.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, 510060, Guangdong, China.

Purpose: This study aimed to determine the susceptibility and the changes of bacterial agents of chronic dacryocystitis and determine the risk factors for bacterial prevalence and drug sensitivity to provide a reference for clinical selection of antibiotics.

Methods: A case-control study was conducted using 112 patients with chronic dacryocystitis and 112 patients with non-infectious ophthalmopathy between August 2017 and April 2018. Lacrimal and conjunctival sac secretions were cultured for aerobic and anaerobic bacteria. Forty-five patients with chronic dacryocystitis between November 2014 and November 2015 were also included.

Results: Positive bacterial cultures were obtained from 61.9% and 50.9% of chronic dacryocystitis and non-infectious ophthalmopathy patients, but the detection rates for pathogenic bacteria were 18.3% and 2.7%, respectively (P > 0.001). Gram-negative and anaerobic bacteria were significantly more prevalent in the patient group compared with the control group (P = 0.001 and 0.005, respectively). Bacteria were detected at a significantly higher rate in patients with irritant symptoms (itch or foreign-body sensation) than in those without (OR = 9.333, P = 0.002), particularly Staphylococcus (OR = 9.783, P = 0.002). 11.6% (10/86) and 55.8% (48/86) showed resistance to levofloxacin and tobramycin, respectively. Compared with three years ago, the detection rate for Gram-positive cocci decreased from 51.1% to 27.8% (χ = 8.054, P = 0.005) CONCLUSIONS: Gram-positive cocci, Gram-negative bacilli, and anaerobic bacteria were the predominant pathogens. The prevalence of Gram-positive bacteria in cases of chronic dacryocystitis is decreasing.
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http://dx.doi.org/10.1007/s10792-020-01545-8DOI Listing
January 2021

The Key Role of VEGF in the Cross Talk between Pterygium and Dry Eye and Its Clinical Significance.

Ophthalmic Res 2020 10;63(3):320-331. Epub 2020 Jan 10.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Purpose: To examine whether dry eye severity is a risk factor for pterygium activity and whether vascular endothelial growth factor (VEGF) is crucial in the cross talk between pterygium and dry eye.

Methods: A total of 103 patients with primary pterygium (Pteg) were included in the study group; they were divided into 2 groups according to the complication of dry eye (DE) (Pteg + DE group, Pteg - DE group). Further, 60 patients with just dry eye (DE group) and 60 normal individuals (normal) were included as 2 control groups. DE severity and pterygium activity were measured, and unstimulated tear samples and pterygium tissues were collected for cytokine detection.

Results: (1) Tear detection: VEGF expression increased in the Pteg + DE group compared to the Pteg - DE, DE, and normal control groups; VEGF was especially increased in the active Pteg + DE group. VEGF concentration was positively correlated with pterygium activity. (2) Tissue detection: the mRNA expression of VEGF was upregulated in the active pterygium group.

Conclusions: Inflammation played an important role in the development of dry eye and pterygium. VEGF was the core molecule in the cross talk, which might explain the high incidence of the coexistence of these 2 diseases.
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http://dx.doi.org/10.1159/000503636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7257259PMC
March 2021

Treatment with solubilized Silk-Derived Protein (SDP) enhances rabbit corneal epithelial wound healing.

PLoS One 2017 20;12(11):e0188154. Epub 2017 Nov 20.

Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

There is a significant clinical need to improve current therapeutic approaches to treat ocular surface injuries and disease, which affect hundreds of millions of people annually worldwide. The work presented here demonstrates that the presence of Silk-Derived Protein (SDP) on the healing rabbit corneal surface, administered in an eye drop formulation, corresponds with an enhanced epithelial wound healing profile. Rabbit corneas were denuded of their epithelial surface, and then treated for 72-hours with either PBS or PBS containing 5 or 20 mg/mL SDP in solution four times per day. Post-injury treatment with SDP formulations was found to accelerate the acute healing phase of the injured rabbit corneal epithelium. In addition, the use of SDP corresponded with an enhanced tissue healing profile through the formation of a multi-layered epithelial surface with increased tight junction formation. Additional biological effects were also revealed that included increased epithelial proliferation, and increased focal adhesion formation with a corresponding reduction in the presence of MMP-9 enzyme. These in vivo findings demonstrate for the first time that the presence of SDP on the injured ocular surface may aid to improve various steps of rabbit corneal wound healing, and provides evidence that SDP may have applicability as an ingredient in therapeutic ophthalmic formulations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188154PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695843PMC
December 2017

Corticosteroids effects on LPS-induced rat inflammatory keratocyte cell model.

PLoS One 2017 27;12(4):e0176639. Epub 2017 Apr 27.

Ophthalmology Department, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

Purpose: Corticosteroids are efficient anti-inflammation treatments. However, there are still arguments on whether it should be used in keratitis. This study was to observe the effect of corticosteroids on keratocytes both in normal condition and inflammation status in vitro.

Methods: Rat keratocytes were cultured and used for examination. 10 μg/ml lipopolysaccharide (LPS) was used to establish the inflammatory keratocyte cell model, and prednisolone acetate (PA), dexamethasone (Dex) and fluorometholone (Flu) were used as corticosteroids treatments. 5 d-growth curve and cell viabilities were assayed by CCK8, and cell morphologies and migration rate were studied. TNF-α, IL-6 and IL-1β levels were examined by ELISA. Western blotting was used to quantified type VI collagen (Col VI) and matrix metalloproteinase 9 (MMP9) expressions, and immunofluorescence staining assays of Col I and Col VI were carried out.

Results: In normal condition, proliferation and migration of keratocytes were slightly influenced in PA, Dex and Flu groups. The secretion of Col I and Col VI was suppressed and MMP9 expression increased in corticosteroids groups. But no significant difference was seen in TNF-α, IL-6 and IL-1β expression levels. In inflammatory status, TNF-α, IL-6 and MMP9 levels increased in LPS group, while they significantly decreased in corticosteroids groups. Although keratocytes viabilities and migration were slightly affected in 24 h, no significant differences were seen between LPS group and corticosteroids groups in 5-d proliferation. Col I and Col VI secretion in LPS-keratocytes was maintained with corticosteroids treatments.

Conclusions: Corticosteroids showed lightly effects on keratocytes proliferation and migration, but it successfully decreased TNF-α, IL-6 level and maintained the secretion of and Col I and Col VI, while suppressed the expression of MMP9 in LPS-induced keratocytes. PA was suggested to use in early stage of keratitis clinical treatment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176639PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407809PMC
September 2017

Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation.

Invest Ophthalmol Vis Sci 2017 03;58(3):1425-1433

Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States.

Purpose: The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin-derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model.

Methods: Solubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies.

Results: Results indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls.

Conclusions: The addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.
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http://dx.doi.org/10.1167/iovs.16-19957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022413PMC
March 2017

LIM Homeobox Domain 2 Is Required for Corneal Epithelial Homeostasis.

Stem Cells 2016 Feb 5;34(2):493-503. Epub 2016 Jan 5.

Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, Illinois, USA.

The cornea requires constant epithelial renewal to maintain clarity for appropriate vision. A subset of stem cells residing at the limbus is primarily responsible for maintaining corneal epithelium homeostasis. Trauma and disease may lead to stem cell deficiency and therapeutic targeting to replenish the stemness capacity has been stalled by the lack of reliable corneal epithelial stem cell markers. Here we identified the location of Lhx2 in mice (mLhx2) cornea and conjunctival tissue using an Lhx2eGFP reporter model and in human tissues (hLHX2). Lhx2 localized to the basal cells of central cornea, the conjunctiva and the entire limbal epithelium in humans and mice. To ascribe a functional role we generated Lhx2 conditional knockout (cKO) mice and the phenotypic effects in corneas were analyzed by slit lamp microscopy, in cell-based assays and in a model of corneal epithelium debridement. Immunodetection on corneal sections were used to visualize conjunctivalization, a sign of limbal barrier failure. Lhx2cKO mice produced reduced body hair and spontaneous epithelial defects in the cornea that included neovascularization, perforation with formation of scar tissue and opacification. Cell based assays showed that Lhx2cKO derived corneal epithelial cells have a significantly lower capacity to form colonies over time and delayed wound-healing recovery when compared to wildtype cells. Repeated corneal epithelial wounding resulted in decreased re-epithelialization and multiple cornea lesions in Lhx2cKO mice compared to normal recovery seen in wildtype mice. We conclude that Lhx2 is required for maintenance of the corneal epithelial cell compartment and the limbal barrier.
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http://dx.doi.org/10.1002/stem.2257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4834794PMC
February 2016

Impaired systemic vascular endothelial function in patients with non-arteritic anterior ischaemic optic neuropathy.

Graefes Arch Clin Exp Ophthalmol 2016 May 5;254(5):977-81. Epub 2015 Nov 5.

Department of Ophthalmology, The First Affiliated Hospital of Sun Yat-sen University, #58 Zhongshan Second Road, Guangzhou, Guangdong, China, 510080.

Purpose: The purpose of this study was to evaluate systemic endothelial function in elderly hypertension patients with non-arteritic anterior ischaemic optic neuropathy (NAION) by using a noninvasive physiological method: endothelium-dependent, flow-mediated vasodilation (FMD).

Methods: Forty-two systemic hypertension patients with NAION (NAION group), 64 age- and sex-matched patients with systemic hypertension and no other ocular disease (hypertension group), and 100 age- and sex-matched healthy volunteers (normal group) were enrolled. FMD was evaluated using a high-resolution ultrasonography. Traditional cardiovascular risk factors and vascular parameters were measured.

Results: Systolic blood pressure and diastolic blood pressure were significantly higher in patients with NAION compared with the control groups (p < 0.001). The FMD decreased significantly in the NAION group (6.02 ± 1.87 %) compared to in the hypertension group (7.86 ± 2.94 %, p < 0.001) and the normal group (8.99 ± 2.44 %, p < 0.001). By multivariable logistic regression analysis, FMD was significantly associated with NAION (OR, 1.79; 95%CI, 1.67-2.01).

Conclusions: NAION may be associated with systemic vascular endothelial dysfunction. FMD might be useful in the treatment monitoring of NAION.
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http://dx.doi.org/10.1007/s00417-015-3212-yDOI Listing
May 2016

Reconstruction of Highly Proliferative Auto-Tissue-Engineered Lamellar Cornea Enhanced by Embryonic Stem Cell.

Tissue Eng Part C Methods 2015 Jul 20;21(7):639-48. Epub 2015 Jan 20.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University , Guangzhou, China .

To increase the epithelial proliferation of an auto-tissue-engineered lamellar cornea, 3.0 × 10(6) corneal epithelial cells (CECs) were combined with 3 × 10(5) mouse embryonic stem cells (ESCs) pretransfected with the HSV-tk gene (CECs + ESCs-TK group), and 3.3×10(6) corneal epithelial cells (CECs group) were seeded between the acellular porcine corneal stroma and the amniotic membrane using the centrifugal cell seeding method. After 4 days of perfusion culture (treatment with ganciclovir starting on day 2), a thicker corneal epithelium (four to five layers) formed in the CECs + ESCs-TK group compared with that observed in the CECs group (two to three layers). More stem/progenitor cell (K3 -, p63+, ABCG2+, and integrin-β1+) and proliferation phenotypes (Ki67+) were measured in the CECs + ESCs-TK group compared with the CECs group using immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, and flow cytometry. Consistent with these findings, the colony-forming efficiency and cellular doubling time were significantly different between the CECs + ESCs-TK group (16.18% ± 3.98%, 28.45 ± 2.03 h) and CECs group (11.96% ± 2.60%, 36.3 ± 1.15 h). In a rabbit lamellar transplantation model, the CECs + ESCs-TK group had better epithelial barrier functions and wound healing abilities compared with the CECs group. Furthermore, ESCs-TK could be completely and safely removed by ganciclovir. Thus, the ESCS-TK coculture system could serve as a potential strategy for corneal tissue engineering.
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http://dx.doi.org/10.1089/ten.TEC.2014.0481DOI Listing
July 2015

Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

Cell Tissue Res 2015 Feb 15;359(2):547-563. Epub 2014 Nov 15.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xian Lie Nan Road, Guangzhou, 510060, People's Republic of China.

Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.
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http://dx.doi.org/10.1007/s00441-014-2032-4DOI Listing
February 2015

Safety and efficacy of embryonic stem cell microenvironment in a leukemia mouse model.

Stem Cells Dev 2014 Aug 10;23(15):1741-54. Epub 2014 Jun 10.

1 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University , Guangzhou, People's Republic of China .

The embryonic stem cell (ESC) microenvironment can promote the proliferation of terminal cells and reduce the invasiveness of tumor cells. However, implanting ESCs directly in vivo can result in tumorigenicity, immune rejection after differentiation, and graft-versus-host reaction. Therefore, safety is very important in the clinical application of ESCs. We injected ESCs modified with a suicide gene into a leukemia mouse model through peripheral blood to observe the treatment effectiveness. In addition, according to the pre-test, we set the time point of differentiation after transplantation and then activated the suicide gene to kill the ESCs that we had initially implanted, hoping to avoid the risks mentioned earlier. Our results indicated that the body weight and survival rates of mice treated with an ESC microenvironment increased, and leukemic cells in peripheral blood and bone marrow decreased compared with untreated mice. There was no obvious teratoma in mice that received ESC therapy and induced the suicide gene at the proper time during the observation period, while an apparent teratoma was observed in the lungs of mice which received ESC therapy and never induced the suicide gene. Therefore, the ESC microenvironment can promote self-healing of the in vivo microenvironment. Inducing the suicide gene at the appropriate time can reduce or even avoid tumorigenicity and immune rejection after transplantation of ESCs in vivo and improve the safety of their clinical application.
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http://dx.doi.org/10.1089/scd.2013.0585DOI Listing
August 2014

Reconstruction of auto-tissue-engineered lamellar cornea by dynamic culture for transplantation: a rabbit model.

PLoS One 2014 4;9(4):e93012. Epub 2014 Apr 4.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

To construct an auto-tissue-engineered lamellar cornea (ATELC) for transplantation, based on acellular porcine corneal stroma and autologous corneal limbal explants, a dynamic culture process, which composed of a submersion culture, a perfusion culture and a dynamic air-liquid interface culture, was performed using appropriate parameters. The results showed that the ATELC-Dynamic possessed histological structure and DNA content that were similar to native lamellar cornea (NLC, p>0.05). Compared to NLC, the protein contents of zonula occludens-1, desmocollin-2 and integrin β4 in ATELC-Dynamic reached 93%, 89% and 73%, respectively. The basal cells of ATELC-Dynamic showed a better differentiation phenotype (K3-, P63+, ABCG2+) compared with that of ATELC in static air-lift culture (ATELC-Static, K3+, P63-, ABCG2-). Accordingly, the cell-cloning efficiency of ATELC-Dynamic (9.72±3.5%) was significantly higher than that of ATELC-Static (2.13±1.46%, p<0.05). The levels of trans-epithelial electrical resistance, light transmittance and areal modulus variation in ATELC-Dynamic all reached those of NLC (p>0.05). Rabbit lamellar keratoplasty showed that the barrier function of ATELC-Dynamic was intact, and there were no signs of epithelial shedding or neovascularization. Furthermore, the ATELC-Dynamic group had similar optical properties and wound healing processes compared with the NLC group. Thus, the sequential dynamic culture process that was designed according to corneal physiological characteristics could successfully reconstruct an auto-lamellar cornea with favorable morphological characteristics and satisfactory physiological function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093012PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976280PMC
June 2015

ES micro-environment enhances stemness and inhibits apoptosis in human limbal stem cells via the maintenance of telomerase activity.

PLoS One 2013 11;8(1):e53576. Epub 2013 Jan 11.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong, China.

Our previous work had found that telomerase rejuvenated in the cytoplasm of corneal epithelial cells cultured in embryonic stem cell-conditioned medium, the functional properties of stem-like corneal epithelial cells can be enhanced by co-culturing with embryonic stem cells (ESCs) via activation of the integrinβ1-FAK-PI3K/Akt signaling pathway. The goal of this study was to explore the potential molecular mechanisms of the ES micro-environment that enhance the stem cell-like phenotype and inhibit apoptosis in human limbal stem cells (LSC). The LSC were cultured in different media, either CnT-20 medium or CnT-20 +20% ES culture supernatant (ESC-CM). We observed that LSC cultured in ESC-CM had an increased proliferative capacity, greater serial passage capacity, higher colony-forming efficiency (CFE) and higher levels of stem cell-associated marker than those cultured in CnT-20. Compared with CnT-20, ESC-CM enhanced the undifferentiated status and inhibited apoptosis in the LSC by promoting the maintenance of telomerase activity, which could reduce the generation of reactive oxygen species (ROS), maintain the membrane potential (Δψm) at higher levels and reduce the expression of the p21 protein. Our findings indicated that ESC-CM system induced LSC to maintain a stem cell phenotype and inhibit the process of apoptosis. These effects might partially be achieved via the telomerase-p21-mitochondrial axis and the activation of the FAK/Wnt signaling pathways. This study may have high impact and clinic implication on the expansion of LSC in regenerative medicine, especially for ocular surface reconstruction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053576PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543452PMC
July 2013

Comparative study of the effects of recombinant human epidermal growth factor and basic fibroblast growth factor on corneal epithelial wound healing and neovascularization in vivo and in vitro.

Ophthalmic Res 2013 18;49(3):150-60. Epub 2012 Dec 18.

Department of Ophthalmology, Zhujiang Hospital, Southern Medical University, Guangzhou, PR China.

Purpose: This study was undertaken to investigate the effects of recombinant human epidermal growth factor (rhEGF) and basic fibroblast growth factor (bFGF) on corneal wound healing and neovascularization (CNV).

Methods: The positive effects of 10 ng/ml rhEGF and bFGF on the proliferation of corneal epithelial cells (SD-HCEC1s), rabbit keratocyte cells (RKCs) and human umbilical vein endothelial cells (HUVECs) as well as the effects on the migration capacity on HUVECs were observed. An animal central corneal wound and CNV model was established in rabbits. One eye of each group was chosen randomly for topical administration of rhEGF, bFGF or normal saline, and variability in the area of corneal epithelial wound healing and CNV was observed.

Results: The optimal concentration of rhEGF and bFGF for the proliferation of corneal epithelial cells was 10 ng/ml. The promotive effect of 10 ng/ml rhEGF on the proliferation of RKCs and HUVECs was less than that of 10 ng/ml bFGF. In the animal experiment, the healing rate of the corneal epithelium in the rhEGF group was better than in the other groups on day 1. On day 3, the healing rates of the 3 groups were nearly equal. The CNV area in the rhEGF group was less than that of the bFGF group.

Conclusions: rhEGF and bFGF both had promotive effects on corneal epithelial wound healing, but rhEGF had a weaker promotive effect on CNV than bFGF. With long-term application of growth factor drugs, rhEGF is suggested for lessening the growth of CNV.
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http://dx.doi.org/10.1159/000343775DOI Listing
September 2013

Using genipin-crosslinked acellular porcine corneal stroma for cosmetic corneal lens implants.

Biomaterials 2012 Oct 15;33(30):7336-46. Epub 2012 Jul 15.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, PR China.

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4 h at 37 °C, followed by lyophilization at -10 °C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7 ± 4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7 ± 4.6% and 23.01 ± 3.45 m(2)/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5 min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.
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http://dx.doi.org/10.1016/j.biomaterials.2012.06.080DOI Listing
October 2012

Long-term cultivation of human corneal endothelial cells by telomerase expression.

Exp Eye Res 2012 Jul 30;100:40-51. Epub 2012 Apr 30.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.

The objective of this study was to explore the potential role of human telomerase reverse transcriptase (TERT) in extending the proliferative lifespan of human corneal endothelial cells (HCECs) under long-term cultivation. A primary culture was initiated with a pure population of HCECs in DMEM/F12 media containing 10% fetal bovine serum and other various supplements. TERT gene was successfully transfected into normal HCECs. A stable HCECs cell line (TERT-HCECs) that expressed TERT was established. The cells could be subcultured for 36 passages. Within this line of cells, TERT not only extended proliferative lifespan and inhibited apoptosis but also enhanced the cell line remaining the normal characteristics similar to HCECs. There were no significantly differences in the expression of the pump function related proteins voltage dependent anion channel 3 (VDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), Na(+)/K(+)-ATPase α1, and ZO-1 in the cell line TERT-HCECs and primary HCECs. TERT-HCECs formed a monolayer cell sheet, maintained similar cell junction formation and pump function with primary HCECs. Karyotype analysis exhibited normal chromosomal numbers. The soft agar colony assay and tumor formation in nude mice assay showed no malignant alterations in TERT-HCECs. Our findings indicated that we had established a cell line with its similar phenotype and properties to primary HCECs. Further study of the TERT-HCECs may be valuable in studying the function of the cells in vivo.
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http://dx.doi.org/10.1016/j.exer.2012.04.013DOI Listing
July 2012

Research on the stability of a rabbit dry eye model induced by topical application of the preservative benzalkonium chloride.

PLoS One 2012 16;7(3):e33688. Epub 2012 Mar 16.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Background: Dry eye is a common disease worldwide, and animal models are critical for the study of it. At present, there is no research about the stability of the extant animal models, which may have negative implications for previous dry eye studies. In this study, we observed the stability of a rabbit dry eye model induced by the topical benzalkonium chloride (BAC) and determined the valid time of this model.

Methods And Findings: Eighty white rabbits were randomly divided into four groups. One eye from each rabbit was randomly chosen to receive topical 0.1% BAC twice daily for 2 weeks (Group BAC-W2), 3 weeks (Group BAC-W3), 4 weeks (Group BAC-W4), or 5 weeks (Group BAC-W5). Fluorescein staining, Schirmer's tests, and conjunctival impression cytology were performed before BAC treatment (normal) and on days 0, 7, 14 and 21 after BAC removal. The eyeballs were collected at these time points for immunofluorescence staining, hematoxylin and eosin (HE) staining, and electron microscopy. After removing BAC, the signs of dry eye in Group BAC-W2 lasted one week. Compared with normal, there were still significant differences in the results of Schirmer's tests and fluorescein staining in Groups BAC-W3 and BAC-W4 on day 7 (P<0.05) and in Group BAC-W5 on day 14 (P<0.05). Decreases in goblet cell density remained stable in the three experimental groups at all time points (P<0.001). Decreased levels of mucin-5 subtype AC (MUC5AC), along with histopathological and ultrastructural disorders of the cornea and conjunctiva could be observed in Group BAC-W4 and particularly in Group BAC-W5 until day 21.

Conclusions: A stable rabbit dry eye model was induced by topical 0.1% BAC for 5 weeks, and after BAC removal, the signs of dry eye were sustained for 2 weeks (for the mixed type of dry eye) or for at least 3 weeks (for mucin-deficient dry eye).
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033688PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306287PMC
August 2012

Tectonic lamellar keratoplasty with acellular corneal stroma in high-risk corneal transplantation.

Mol Vis 2011 15;17:1909-17. Epub 2011 Jul 15.

State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China.

Purpose: Tectonic lamellar keratoplasty (TLKP) is a primary surgical procedure to improve the condition of the recipient bed in high-risk corneal transplantation. It is usually performed for a secondary optical penetrating keratoplasty (PKP). The present study was undertaken to explore a new strategy for TLKP using acellular corneal stroma (ACS) prepared by decellularization.

Methods: ACS for TLKP was prepared from cat cornea by decellularization. The efficiency of the decellularization was examined by hematoxylin and eosin (H&E) staining and through DNA content analysis. Twenty-eight New Zealand white rabbits, as recipients, were assigned to one of two groups that had different material for their TLKP. The TLKP was combined with a central optical PKP as a single-stage procedure. Either ACS or fresh cat corneal lamella, 11.25 mm in diameter, was used for the TLKP in these two groups. After TLKP, a 6.5-mm full-thickness cat cornea was placed in the central cornea of each recipient rabbit for PKP. Clinical outcomes and the histology of the transplants were compared post-operatively.

Results: ACS for TLKP prolonged the survival of the transplants. The mean survival time of the transplants in the ACS group (36.4±4.3 days) was longer than for those in the control group (14.0±2.2 days, p<0.05). The ACS group showed a significantly smaller neovascularization area compared to the control group. The areas of corneal neovascularization were 5.3±1.1 mm² and 45.2±4.9 mm² (p<0.05), respectively, after two weeks, and 25.1±4.7 mm² and 105.3±12.4 mm² (p<0.05), respectively, after four weeks. Histology revealed that fewer inflammatory cells were infiltrating the transplants in the ACS group than those in the control group.

Conclusions: The use of ACS for TLKP prolonged the survival of corneal transplants, reduced corneal neovascularization, and prevented from infiltration of inflammatory cells. It is a feasible and effective strategy to prolong the survival of transplants in high-risk corneal transplantation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154122PMC
December 2011

Using acellular porcine limbal stroma for rabbit limbal stem cell microenvironment reconstruction.

Biomaterials 2011 Nov 23;32(31):7812-21. Epub 2011 Jul 23.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xian Lie Nan Road, Guangzhou 510060, PR China.

To investigate the feasibility of using acellular porcine limbal stroma for limbal stem cell microenvironment reconstruction. Limbal reconstruction was performed in rabbit partial limbal defect models. Rabbits were randomly divided into four groups: acellular porcine limbal stroma, de-epithelized rabbit limbal autograft stroma, de-epithelized porcine limbal stroma and acellular porcine corneal stroma transplantation groups. In both the acellular porcine limbal stroma and de-epithelized rabbit limbal autograft stroma groups, cornea transparency and epithelium integrity were sustained and graft rejection was not observed. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1, but it returned to K3-/P63+/Ki67+(phenotype characteristic of limbal epithelium) by postoperative months 3 and 6. In the de-epithelized porcine limbal stroma group, acute and serious immune rejection occurred by postoperative days 8-10. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative month 1. In the acellular porcine corneal stroma group, there were some new vessel invasion into the peripheral cornea and mild corneal opacity. The basal epithelial cells of the grafts showed the K3+/P63+/Ki67+ phenotype at postoperative months 1, 3, and 6. In conclusion, acellular porcine limbal stroma possessed very low immunogenicity, retained a good original limbal ECM microenvironment, and thus the reconstructed rabbit limbal microenvironment maintained limbal epithelial stem cell stemness and proliferation.
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http://dx.doi.org/10.1016/j.biomaterials.2011.07.012DOI Listing
November 2011

Comparison of clinical outcomes between iris-fixated anterior chamber intraocular lenses and scleral-fixated posterior chamber intraocular lenses in Marfan syndrome with lens subluxation.

Clin Exp Ophthalmol 2012 Apr 7;40(3):268-74. Epub 2011 Aug 7.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Background: To compare the clinical outcomes in Marfan's with subluxated lens having phaco-emulsification with simultaneous scleral-fixated posterior chamber intraocular lens or iris-fixated anterior chamber intraocular lens implantation.

Design: Randomized case series in the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Participants: Seventy-one eyes of 49 patients with Marfan syndrome with subluxated lens.

Methods: This is a randomized case series of patients with Marfan syndrome and subluxated lenses who underwent phaco-emulsification combined with scleral-fixated posterior chamber intraocular lens or iris-fixated anterior chamber intraocular lens implantation.

Main Outcome Measures: The evaluation indexes included the surgery time, best corrected visual acuity, intraocular pressure, aqueous flare and cells counts, corneal endothelium counts and complications.

Results: Increase in best corrected visual acuity in both groups was not significant. The aqueous flare and cells rose in both groups postoperatively. Significant difference between the two groups at 1 week postoperatively was found, whereas no statistically significant difference was found later. The loss rate of corneal endothelium cells in the scleral-fixated posterior chamber intraocular lens group was 13.2% and 19.5% at 3 months and 1 year postoperatively, which in the iris-fixated anterior chamber intraocular lens group was 13.3% and 19.3% (P > 0.05). Prolapse of vitreous was found in 21 cases intraoperatively. The posterior capsule opacification rate was 32% and 15%, respectively. The decentration of the intraocular lens was found in 19 eyes (48.7%) in the scleral-fixated posterior chamber intraocular lens group 1 year postoperatively, whereas none was found in the iris-fixated anterior chamber intraocular lens group.

Conclusions: Iris-fixated anterior chamber intraocular lens after phaco-emulsification presented a safe, simple and efficient approach for managing subluxated lens in Marfan syndrome.
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http://dx.doi.org/10.1111/j.1442-9071.2011.02612.xDOI Listing
April 2012

A novel procedure for treating canalicular obstruction by re-canaliculisation and bicanalicular intubation.

Br J Ophthalmol 2012 Mar 7;96(3):366-9. Epub 2011 Jun 7.

Department of ENT, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, The People's Republic of China.

Aim: The aim of this study was to evaluate a new procedure for treating canalicular obstruction by re-canaliculisation and bicanalicular intubation (RC-BCI).

Methods: Thirty adult patients (32 eyes) with canalicular obstruction were treated with RC-BCI from September 2005 to December 2007 at Zhongshan Ophthalmic Centre (Guangzhou, China). Silicone tubes were left in place for 2-3 months and were removed when patients had relief by tearing. Patients were evaluated postoperatively by symptoms, lacrimal irrigation and satisfaction rate.

Results: Mean follow-up time after tube removal was 21.5 (range 6-26) months. Twenty-six eyes (81.25%) had complete epiphora relief, two eyes (6.25%) had partial relief and four eyes (12.5%) had no improvement after the removal of the tubes. One eye (3.13%) had lower punctum splitting 2 months after the surgery. The overall satisfaction rate was 93.3% in 30 patients. No other complications occurred.

Conclusion: Our findings demonstrated that the RC-BCI was an effective procedure for treating canalicular obstruction with few complications.
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http://dx.doi.org/10.1136/bjo.2011.202200DOI Listing
March 2012

Enhanced functional properties of corneal epithelial cells by coculture with embryonic stem cells via the integrin β1-FAK-PI3K/Akt pathway.

Int J Biochem Cell Biol 2011 Aug 28;43(8):1168-77. Epub 2011 Apr 28.

State Key Laboratory of Ophthalmology, Sun yet-sen University, Guangzhou, Guangdong, PR China.

Adult stem cells are important cell sources in regenerative medicine, but isolating them is technically challenging. This study employed a novel strategy to generate stem-like corneal epithelial cells and promote the functional properties of these cells by coculture with embryonic stem cells. The primary corneal epithelial cells were labelled with GFP and cocultured with embryonic stem cells in a transwell or by direct cell-cell contact. The embryonic stem cells were pre-transfected with HSV-tk-puro plasmids and became sensitive to ganciclovir. After 10 days of coculture, the corneal epithelial cells were isolated by treating the cultures with ganciclovir to kill the embryonic stem cells. The expression of stem cell-associated markers (ABCG2, p63) increased whereas the differentiation mark (Keratin 3) decreased in corneal epithelial cells isolated from the cocultures as evaluated by RT-PCR and flow cytometry. Their functional properties of corneal epithelial cells, including cell adhesion, migration and proliferation, were also enhanced. These cells could regenerate a functional stratified corneal epithelial equivalent but did not form tumors. Integrin β1, phosphorylated focal adhesion kinase and Akt were significantly upregulated in corneal epithelial cells. FAK Inhibitor 14 that suppressed the expression of phosphorylated focal adhesion kinase and Akt inhibited cell adhesion, migration and proliferation. LY294002 that suppressed phosphorylated Akt but not phosphorylated focal adhesion kinase inhibited cell proliferation but had no effect on cell adhesion or migration. These findings demonstrated that the functional properties of stem-like corneal epithelial cells were enhanced by cocultured embryonic stem cells via activation of the integrin β1-FAK-PI3K/Akt signalling pathway.
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http://dx.doi.org/10.1016/j.biocel.2011.04.010DOI Listing
August 2011

Rapidly constructed scaffold-free cornea epithelial sheets for ocular surface reconstruction.

Tissue Eng Part C Methods 2011 May 14;17(5):569-77. Epub 2011 Feb 14.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, PR China.

Purpose: To develop a centrifugal cell seeding method for rapid and efficient reconstruction of ocular surface with limbal stem cell deficiency (LSCD) in rabbits.

Methods: The orthogonal design method was used to optimize centrifugation parameters for cell seeding. Methylthiazol tetrazolium proliferation assay, colony-forming efficiency, and flow cytometry were used to study cell viability. Histology, electron microscopy, and immunocytochemistry were evaluated for centrifugation-constructed cornea epithelial sheets (CCCESs). The rabbit eyes with LSCD were treated with or without CCCES for in vivo evaluation.

Results: The 80.04% attached cells with 98.04% viability were achieved using optimal cell seeding density at 9 × 10(5) cm(-2) with centrifugation at 1800 rpm for 4 min. The 0.4% glycerin was added in the medium to increase the surface tension and osmotic pressure to optimal condition for obtaining higher cell density. The three-layer epithelial sheets were rapid constructed, which displayed the characteristics of normal corneal epithelium. In vivo transplantation, labeled cells of CCCES were detected at 30 days. CCCES reconstructed the LSCD corneal epithelia without conjunctivalization and neovascularation, evidenced by positive K3 and negative K4, Muc5AC.

Conclusion: The scaffold-free corneal epithelial sheets were rapidly constructed using optimal centrifugation procedure, which was demonstrated to reconstruct ocular surface with LSCD.
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http://dx.doi.org/10.1089/ten.TEC.2010.0529DOI Listing
May 2011

Cell delivery with fixed amniotic membrane reconstructs corneal epithelium in rabbits with limbal stem cell deficiency.

Invest Ophthalmol Vis Sci 2011 Feb 9;52(2):724-30. Epub 2011 Feb 9.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Peoples Republic of China.

Purpose: To explore the feasibility and efficacy of a cell delivery system using amniotic membrane (AM) fixed by a novel biomembrane-fixing device (BMFD) for corneal epithelium reconstruction in rabbits with limbal stem cell deficiency (LSCD).

Methods: Sixty female rabbits with LSCD were created and randomly assigned to three groups of 20 each: LSCD rabbits without treatment (the control), LSCD rabbits treated with BMFD-fixed AM (BMFD-AM), and rabbits treated with male human limbal epithelial cells delivered with BMFD-fixed AM (BMFD-AM+cells). They were followed up with slit lamp observation and corneal fluorescein staining for 14 days. Cytokeratin K3 and K4 and mucin 5AC were used to evaluate corneal conjunctivalization. Sry gene detection was used to trace the delivered cells.

Results: The corneal re-epithelialization time was 5.60 ± 0.46 days in the BMFD-AM+cell group, significantly shorter (P < 0.05) than in the LSCD (12.45 ± 0.65 days) and the BMFD-AM (9.25 ± 0.51 days) groups. Conjunctivalization and neovascularization were observed to be severe in the LSCD group and moderate in the BMFD-AM group. The prevention of conjunctivalization in the BMFD-AM+cell group was evidenced by positive K3/K12 and negative MUC5AC and K4 observed on re-epithelialized corneal epithelium. The histologic sections at different time points and positive Sry gene expression indicated that the delivered cells adhered to the wounded corneal surface and proliferated well.

Conclusions: These findings demonstrate that the BMFD with fixed AM served well as a cell delivery system for the ocular surface. The delivered limbal epithelial cells promoted corneal re-epithelialization and prevented corneas from conjunctivalization and neovascularization in rabbits with experimental LSCD.
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http://dx.doi.org/10.1167/iovs.10-5291DOI Listing
February 2011

Development and characterization of acellular porcine corneal matrix using sodium dodecylsulfate.

Cornea 2011 Jan;30(1):73-82

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Purpose: The objective of this study was to produce a porcine corneal acellular matrix (ACM) and assess its possibility for biomedical applications.

Methods: Porcine corneas were treated with various concentrations of sodium dodecylsulfate for different lengths of time. Optimal conditions for processing the ACM were noted regarding removal of all cellular components and retention of the spatial arrangement of the corneal stroma. The physical characteristics (including water absorption and light transmittance), biomechanics, and cytotoxicity of the ACM were also found to be conserved. Subsequently, ACM was transplanted into the interlaminar stroma of rabbit corneas. The transparency and structures of the collagen fibers were determined.

Results: By immersing corneal tissues in isotonic buffer containing 0.1% sodium dodecylsulfate for 7 hours, we were able to produce an ACM whose cells were completely removed, without disrupting collagen layer structure. Although water absorption and light transmittance of the ACM decreased when compared with natural corneal stroma, ACM showed similar biomechanical properties and biocompatibility as natural ones. After xenotransplantation into rabbit corneal stromal layers for 4 weeks, both ACM and rabbit corneas showed complete transparency. Almost 1 year postoperatively, the corneas remained transparent with regular stromal structures and ACM appeared stable in situ without deliquescence or immunological rejection.

Conclusions: A simple and valid method to produce decellularized corneal matrix has been successfully developed. These acellular matrices similar to natural corneas in structure, strength, and transparency have tremendous potential for corneal transplantation as ideal implants for donors and for tissue engineering applications as suitable scaffolds.
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http://dx.doi.org/10.1097/ICO.0b013e3181dc8184DOI Listing
January 2011

Carrier-free epithelial cell sheets prepared by enzymatic degradation of collagen gel.

J Tissue Eng Regen Med 2011 Feb;5(2):138-45

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-Sen University, Guangzhou, People's Republic of China.

Limbal stem-cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier-free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier-free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell-cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier-free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration.
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http://dx.doi.org/10.1002/term.298DOI Listing
February 2011

Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium.

Mol Vis 2010 Apr 8;16:611-22. Epub 2010 Apr 8.

State Key laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, PR China.

Purpose: To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro.

Methods: Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet's membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT-PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na(+)-K(+)-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na(+)-K(+)-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT-PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry.

Results: In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3-4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na(+)-K(+)-ATPase, VDAC3, SLC4A4, and CLCN3. The number of Ki67 positive cells, CFE, and percentage of cells entering the S and G(2) phases were higher in the 25%ESC-CM group than in the CEM group. The number of apoptotic cells and p21 protein expression both decreased in the 25%ESC-CM group.

Conclusions: Use of 25%ESC-CM significantly increased the number of proliferating cells. These effects may be achieved through inhibition of p21 expression and apoptosis. These results suggested that 25%ESC-CM may be a new tool for cultivating HCECs for transplantation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2850933PMC
http://dx.doi.org/10.1167/2.7.611DOI Listing
April 2010

Enhancement of long-term proliferative capacity of rabbit corneal epithelial cells by embryonic stem cell conditioned medium.

Tissue Eng Part C Methods 2010 Aug;16(4):793-802

State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, P.R. China.

Induction of autologous stem cells for directed differentiation has become a predominant method to obtain autologous cells for tissue reconstruction. However, the low inducing efficiency and contamination with other type of cells hinder its clinical utilization. Here we report a novel phenomenon that the corneal epithelial cells maintain long-term proliferative capacity and tissue-specific cell phenotype by factors secreted from murine embryonic stem cells (ESCs). The rabbit corneal epithelial cells grew very well in culture medium with addition of 40% ESC conditioned medium (ESC-CM). These corneal epithelial cells have been serially subcultured for more than 20 passages and maintained high cell purity, cobble-stone-like morphology, enhanced colony forming efficiency, normal diploid, and capacity to regenerate a functional stratified corneal epithelial equivalent. More importantly, these cells did not form tumor, and the cells lost their proliferative capacity after withdrawal of ESC-CM. The long-term proliferative capacity of corneal epithelial cells is partly resulted from enhancement of cell survival and colony formation, and mediated by ectopic expression of telomerase. Our findings indicate that this new ESC-CM culture system can generate low-immunogenic autologous cells sufficiently for use in regenerative medicine.
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http://dx.doi.org/10.1089/ten.TEC.2009.0380DOI Listing
August 2010
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